IMMUNOPATHOGENESIS OF UVEITIS AND APPROACHES TO IMMUNOTHERAPY: (1) Previously we demonstrated that autoimmunity to retina could be either Th17 or Th1 driven and that each of these T cell responses was sufficient to mediate disease by itself. Since these two responses, though distinct, are redundant for induction of pathology, we sought an approach that would inhibit both. IL27p28 binds to gp130 and can block both IL-6 and IL-27 signaling, which are involved in Th17 and Th1 responses, respectively. Overexpression of IL27p28 in mice leads to protection from EAU induced by either Th1 or by Th17 cells. This could point to IL-27 and/or its subunits as a therapeutic mechanism for targeting both pathogenic responses. Another question is what are the trigger(s) and source(s) for endogenous production of this cytokine, and whether they can be augmented. We have obtained data suggesting that there is a positive feedback loop between NK cell derived IFN-&#947;acting on DC to produce IL-27, which in turn augments the IFN-&#947;response. This is in line with earlier data published by our lab (Grajewski, Hansen et al., J Immunol 2008) that innate IFN-&#947;production dampens subsequent adaptive IFN-&#947;and IL-17 responses and protects from EAU (Wai Po Chong et al, in preparation). (2) We are continuing to examine the role of IL-22 in the eye. IL-22 is a cytokine produced by Th17 cells and is present in inflammatory sites;however, its effects on the tissue are controversial. It has been reported to have both pro-inflammatory and protective effects, depending on the site and the model. We used IL-22 and IL-22-receptor deficient mice and anti-IL-22 antibodies to examine effects of IL-22 modulation on EAU. Our data do not support a pro-inflammatory role for IL-22 in the eye;rather, IL-22 may have a slight protective role. (Rigden, Mattapallil et al., in preparation). (3) The Gi protein-associated A3 adenosine receptor (A3AR) is highly expressed in inflammatory, but not in normal, lymphoid cells. In collaboration with Drs. Fishman and Bar Yehuda of CanFite Biopharma we have used a small molecule inhibitor of the A3AR, CF101, to modulate EAU starting at the time of disease onset. EAU and its associated inflammatory cytokine responses were ameliorated through a mechanism involving inhibition of PI3K and STAT1 (Bar-Yehuda et al, 2011). EFFECTS OF INNATE IMMUNE RESPONSES ON AUTOIMMUNITY: The innate immune response directly affects immunopathogenic processes and also impacts on adaptive immunity. We previously identified a population of NKT cells that express the IL-23R constitutively and produce IL-17 independently of IL-6 and IL-21 (NKT17) (Rachitskaya, Hansen et al 2008). Recent data indicate that IL-17 production in these cells may involve a unique signaling pathway that bypasses STAT3 under some conditions. Furthermore, have now identified a novel population of non-NKT innate T cells that rapidly produce high amounts of IL-17 upon T cell receptor and IL-23 receptor ligation, similarly to NKT17. These cells lack both CD4 and CD8 expression (double negative = DNT) and express the unique PLZF transcription factor characteristic of innate T cells. We are currently characterizing this cell population with the goal of defining its role in host defense and tissue pathology (A. Hansen, in preparation). NEW MODELS OF UVEITIS IN HUMANIZED MICE: (1) In collaboration with Drs. Warren Strober (NIAID) and Guanxun Meng (Shanghai Pasteur Institute) we are studying uveitis in mice with an inflammasome mutation. These mice are knock-in for a mutated NLRP3 gene associated with Muckle-Wells syndrome, which among its pathologies is also associated with ocular inflammation. Preliminary results show effects on immune responses and histology of EAU and on LPS induced uveitis, models in which inflammasome responses are heavily involved. (2) Immunological responses to S-Ag have been implicated in human uveitis, but direct study of uveitogenic epitopes in humans is not possible. We have established a "humanized" EAU model in HLA transgenic (Tg) mice. Using bioinformatic methods for epitope prediction we are studying recognition and pathogenicity of S-Ag epitopes and have identified several susceptible and resistant HLA alleles. Of particular interest are HLA DR3 Tg mice, which develop disease in response to a region of S Ag recognized by lymphocytes of uveitis patients (peptide M). Together with collaborators from the Benaroya Research Institute we have developed MHC tetramers loaded with peptide M and demonstrated as proof of concept that they detect T lymphocytes in a DR3+ patient with active uveitis. This method could be developed to serve as a biomarker for uveitis (Mattapallil et al., J. Immunol, 2011). (3) Birdshot chorioretinopathy is strongly associated with HLA-A29, but although many patients have responses to S Ag, the precipitating mechanisms are unknown. We have developed HLA-A29 transgenic mice. These mice are developing ocular pathology resembling Birdshot chorioretinopathy within the first few months of life, confirming the involvement of A29 in uveitis. We are characterizing these mice with respect to their visual function and immunological responses, in an attempt to define the mechanisms leading to disease. Preliminary data suggest that A29, rather than serving as an antigen-presenting element, may itself serve as antigen. Projects 2 and 3 above served as a basis for a Bench-to-Bedside application that was awarded for FY 2011 and 2012. FUNDAMENTAL MECHANISMS IN SELF-TOLERANCE AND IN IMMUNE PRIVILEGE: (1) We have made T cell receptor (TCR) Tg mice carrying a TCR specific to the major epitope of IRBP (IRBP TCR Tg), which develop spontaneous EAU disease by 2 months of age that can be very severe. These mice thus represent a new model of spontaneous EAU. They are furthermore serving as a tool to study the development, migration and function of antigen-specific cells in uveitis as well as donors of nave Ag specific T cells for various studies (see ahead). Interestingly, spontaneous uveitis was ameliorated if the IRBP TCR Tg mice are treated with a broad-spectrum antibiotic mixture from before birth, resulting in a drastically altered gut bacterial flora. This suggests that endogenous bacterial flora contributes to development of spontaneous ocular autoimmune disease. (Horai et al, manuscript in preparation). We are currently attempting to dissect which component(s) of the flora is(are) responsible. (2) The eye is an immunologically privileged organ, but paradoxically, remains vulnerable to autoimmunity. Using IRBP specific T cells from the TCR Tg mice described above and FoxP3-GFP reporter mice we re-examined the concept of local immune privilege in vitro and in vivo. Our data show that uncommitted T cells rapidly convert in ocular fluids as well as within the living eye to functional FoxP3+ Tregs in a process involving retinal antigen recognition, de novo FoxP3 induction and proliferation. We examined the molecular mechanisms and demonstrated that this comes at the expense of Th1 and Th2 differentiation and occurs within the eye itself, rather than in draining lymph nodes, suggesting that nave T cells can be primed within the tissue. The inflamed eye is deficient at supporting Treg conversion. Importantly, retina-specific T cells primed in vivo before introduction into the eye were resistant to Treg conversion in the ocular environment, and instead caused severe uveitis. Thus, uncommitted T cells can be disarmed, but immune privilege is unable to protect from uveitogenic T cells that have acquired effector function prior to entering the eye. (Zhou et al, in press, J Immunol 2011 and Zhou et al., submitted).